Lipid peroxidation is a proposed mechanism for the injurious effects of reactive oxygen intermediates and many other toxins. Several methods for measuring lipid peroxidation have been described including measurement of lipofusin, conjugated dienes, aldehydes, pentane, and ethane. These methods are indirect and/or not suitable for in vivo studies. We therefore initiated an attempt to develop more direct analytical procedures for hydroxyfatty acids, which are products of lipid peroxidation, suitable for use in in vivo studies of lipid peroxidation and toxicity. Hydroxyfatty acids were released from phospholipids by enzymatic hydrolysis. The acids were then separated by an HPLC procedure using a reverse-phase chromatography system and were detected and quantitated, against synthesized standards, by UV absorption at 235 nm. This procedure was used successfully to demonstrate the presence of 15-hydroxyeicosatetraenoic acid and 9- and 13-hydroxyoctadecadienoic acids in livers from mice treated in vivo with necrogenic doses of carbon tetrachloride. Livers and lungs from rats given carbon tetrachloride, or from mice given paraquat, showed HPLC peaks indicative of hydroxyfatty acids, although none corresponded exactly with the synthetic standards available. Further studies will continue to probe the development and potential applicability of this methodology to the investigation of the role of lipid peroxidation in tissue injury.